Lecture 5: Bacterial Identification PDF

BACTERIAL IDENTIFICATION 1 LECTURE 5 CREATED BY: Dr RASHA ALREEDY & Dr SULTAN ALSHARIF Edited By: Dr Sarra Rafrafi 2024 2 Identification Methods I. Traditional methods/phenotypic methods II. Immunochemical methods/serological...

BACTERIAL IDENTIFICATION 1 LECTURE 5 CREATED BY: Dr RASHA ALREEDY & Dr SULTAN ALSHARIF Edited By: Dr Sarra Rafrafi 2024 2 Identification Methods I. Traditional methods/phenotypic methods II. Immunochemical methods/serological methods III. Genotypic Methods /Molecular Methods 3 I. PHENOTYPIC METHODS 4 Culture Media For Bacterial Identification The culture media are classified in many different ways: 1. Based on the physical state Liquid media (broth) - Enrichment media Solid media (Agar) Semisolid media 2. Based on nutritional factors Simple or basic media Synthetic media Complex media Special media 5 Types of Culture Media 1. Basic media: Nutrient Agar for most non-fastidious organisms. Other examples: Peptone water and glucose broth. 2. Supportive media: media that contain additional ingredients, such as red blood cells, which support the growth of more fastidious bacteria. 3. Selective media: allow certain types of organisms to grow, and inhibit the growth of other organisms. For example, selective inhibition of some types of microorganisms can be achieved by adding dyes, antibiotics, salts or specific inhibitors which affect the metabolism or enzyme systems of the organisms. 6 NUTRIENT AGAR 7 Types of Culture Media 4. Differential media: media are used to differentiate closely related organisms or groups of organisms due to the presence of certain dyes or chemicals in the media, the organisms will produce characteristic changes or growth patterns that are used for identification or differentiation. E.g. MacConkey agar for Gram(-) bacteria. 5. Cell culture for Chlamydiae, rickettsiae as they are obligate intracellular parasites. 8 MacConkey agar (MAC)  MacConkey agar (MAC) is a bacterial culture medium named after bacteriologist Alfred T. MacConkey (1861-1931).  MacConkey agar is a selective and differentiating agar that only grows gram-negative bacterial species;  It can further differentiate the gram-negative organisms based on their lactose metabolism. 9 PHENOTYPIC CLASSIFICATION  Often does not require sophisticated equipment  Can easily be done anywhere 1. Culture characteristics: colonial morphology. 2. Gram stain (cell wall structure); gram positive or gram negative. 3. Bacterial cell morphology and arrangement 4. ( bacilli, cocci, spiral). 5. Other types of stains; acid fast stain, capsule stain, spore stain, flagella stain….). 10 PHENOTYPIC CLASSIFICATION 6. Growth requirements; oxygen, pH, temperature, and other environmental requirements. 7. Biochemical reactions: enzymes, motility, fermentation…. 8. Nutritional requirement and metabolic capabilities. 9. Resistance or susceptibility to antibacterial agents. 10.Fully or partly automated identification methods (Vitek, Phoenix,..) 11 1. COLONY MORPHOLOGY These are the characteristics used to accurately and consistently describe the morphology of a bacterial colony: Size Shape Texture Height (a.k.a. elevation) Edge (a.k.a. margin) Color (also known as pigmentation) 12 2. MICROSCOPIC PHENOTYPIC EXAM Cell size, shape and arrangement can provide some information for diagnosing certain types of infection. I. Gram stain II. Special stain (e.g. Acid Fast Stain) 13 I. Gram stain  It allows bacteria to be classified as either Gram positive or negative based on their morphology and differential staining properties.  Slides are sequentially stained with crystal violet, iodine, then de-stained with alcohol and counter-stained with safranin.  Gram positive bacteria stain blue-purple and  Gram negative bacteria stain red. 14 15 I. Gram stain  The difference between the two groups is believed to be due to a much greater amount of peptidoglycan in Gram positive cell wall.  As a result the iodine and crystal violet precipitate in the thickened cell wall and are not eluted by alcohol in contrast with the Gram negatives where the crystal violet is readily eluted from the bacteria.  As a result bacteria can be distinguished based on their morphology and staining properties 16 II. Special Stain  Special stains are necessary to bring out characteristics like flagella, capsules, spores and metachromatic granules.  Some bacteria such as mycobacteria (the causative agent of tuberculosis) are not reliably stained due to the large lipid content (Over 60% of the mycobacterial cell wall is lipid mycolic acid and 50% of the dry weight of the cell wall).  Alternative staining techniques (Kinyoun or acid fast stain) are used to stain them  Acid Fast Stain: The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups, particularly the member of genus Mycobacterium. 17 Acid Fast Stain 1. The bacterial smear is stained with carbol fuchsin under heat, it solubilizes the lipoidal material present in the Mycobacterial cell wall. 2. The smear is decolorized (The decolorizing agent is usually acidic alcohol which is usually 3% HCl + 95% ethanol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution. 3. The non-acid fast organism lack the lipoidal material in their cell wall so they are easily decolorized. 18 Acid Fast Stain 4. Then the smear is stained with methylene blue. 5. Only decolorized cells absorb the counter stain and take its color and appears Blue (A) while acid-fast cells retain the Red to Dark pink bacilli (B) color. 19  3. ENVIRONMENTAL REQUIREMENT FOR 20 GROWTH Four most critical factors 1. Oxygen and CO2 availability a) Facultative anaerobic: Some common pathogenic organisms showing the facultative anaerobic mode of respiration. E.coli, Salmonella spp., Staphylococcus aureus …etc b) Strictly aerobic: Pseudomonas spp., Brucella spp., Vibrio Cholerae …etc c) Anaerobic: Peptococcus, Clostridium..etc  3. ENVIRONMENTAL REQUIREMENT FOR 21 GROWTH d) Capnophilic: sometimes referred to as microaerophiles e.g. Neisseria spp., Campylobacter spp., ….etc Carboxyphilic (capnophilic) bacteria; they require an atmosphere composed of oxygen and a raised level of CO2 and therefore grow best in an atmosphere enriched with 5 - 10% CO2 2. Temperature 3. pH-range 4. Moisture  4. NUTRITIONAL REQUIREMENTS AND 22 METABOLIC PATHWAYS A. Single enzyme tests such as: Catalase  Oxidase Indole Urease B. Tests for metabolic pathway(s) such as: Oxidation and fermentation of glucose and other carbohydrates. Amino acid degradation by detection of amino acid decarboxylase. 23 4. NUTRITIONAL REQUIREMENTS AND METABOLIC PATHWAYS C. Establishing inhibitor profiles: The ability to grow in the presence of one or more inhibitory substances can provide valuable identification information. E.g.1 Enterococci can grow in the presence of high NaCl concentration E.g.2 Bacillus spp. can survive ethanol 24 LIMITATION OF PHENOTYPIC METHODS Can not be used if the microbe is not cultivated on an artificial medium known as the viable but non-culturable (VBNC) state, e.g. Treponema Fragility of organisms and failure to survive when transported. Fastidious nature of some microorganisms e.g. Leptospira